, Volume 41, Issue 6, pp 605–614

Accelerated solvent extraction for quantitative measurement of fatty acids in plasma and erythrocytes



Consumption of fish rich in n-3 highly unsaturated FAs (i.e., EPA and DHA) has been suggested to decrease the risk of lifestyle-related diseases such as coronary heart disease, cancer, diabetes, and dementia. Blood eevels of those FA are known appropriate biomarkers of both the corresponding dietary FA intakes and fish consumption. In place of traditional handwork methods for extracting FA, we performed an accelerated solvent extraction (ASE) for at least 13 selected FA in plasma and erythrocytes to measure them by GLC. The FA levels (concentrations and compositions) in 35–50 μL of plasma or erythrocytes were extracted by ASE and measured by GLC. Intra-and interassay coefficients of variation were≤6.0% for both blood materials, except with a minor group of FA (≤1.0% of total FA). When ASE was compared with two traditional handwork methods, FA levels in plasma from 18 healthy subjects were all coincident with very high Pearson's correlation coefficients for the three sets of the sama 18 samples (r≤0.85 to 0.95, P<0.0001), except for 18∶0 (r=0.59, P<0.01). Using ASE and GLC, we have developed a new method for determination the levels of FA in plasma and erythrocytes as biomarkers for dietary intake of fish, fat, and FA. This new method makes it feasible to measure small volumes of samples, automatically, quantitatively, routinely, easily, rapidly and cheaply, with acceptable precision and accuracy.



accelerated solvent extraction


highly unsaturated FA


traditional handwork method


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Egusa, G., Murakami, F., Ito, C., Matsumoto, Y., Kado, S., Okamura, M., Mori, H., Yamane, K., Hara, H., and Yamakido, M. (1993) Westernized Food Habits and Concentrations of Serum Lipids in the Japanese. Arteriosclerosis 100, 249–255.CrossRefGoogle Scholar
  2. 2.
    Hu, F.B., Stampfer, M.J., Manson, J.E., Ascherio, A., Colditz, G.A., Speizer, F.E., Hennekens, C.H., and Willett, W.C. (1999) Dietary Saturated Fats and Their Food Sources in Relation to the Risk of Coronary Heart Disease in Women. Am. J. Clin. Nutr. 70, 1001–1008.PubMedGoogle Scholar
  3. 3.
    World Cancer Research Fond/American Institute for Cancer Research. (1997) Food, Nutrition and the Prevention of Cancer: a Global Perspective, pp. 1–670, AICR, Washington, D.C.Google Scholar
  4. 4.
    Dwyer, J.T. (1997) Human Studies on the Effects of Fatty Acids on Cancer: Summary, Gaps, and Future Research. Am. J. Clin. Nutr. 66 (Suppl 6), 1581S-1586S.PubMedGoogle Scholar
  5. 5.
    Tsunehara, C.H., Leonetti, D.L., and Fujimoto, W.Y. (1990) Diet of Second-Generation Japanese-American Men With and Without Non-Insulin-Dependent Diabetes, Am. J. Clin. Nutr. 52, 731–738.PubMedGoogle Scholar
  6. 6.
    Kalmijn, S., Launer, L.J., Ott, A., Witteman, J.C., Hofman, A., and Breteler, M.M. (1997) Dietary Fat Intake and the Risk of Incident Dementia in the Rotterdam Study, Ann. Neurol. 42, 776–782.PubMedCrossRefGoogle Scholar
  7. 7.
    Kankaanpaa, P., Sutas, Y., Salminen, S., Lichtenstein, A., and Isolauri, E. (1999) Dietary Fatty Acids and Allergy, Ann. Med. 31, 282–287.PubMedGoogle Scholar
  8. 8.
    Dolecek, T.A. (1992) Epidemiological Evidence of Relationships Between Dietary Polyunsaturated Fatty Acids and Mortality in the Multiple Risk Factor Intervention Trial, Proc. Soc. Exp. Biol. Med. 200, 177–182.PubMedGoogle Scholar
  9. 9.
    Zock, P.L., and Katan, M.B. (1998) Linoleic Acid Intake and Cancer Risk: A Review and Meta-Analysis, Am. J. Clin. Nutr. 68, 142–153.PubMedGoogle Scholar
  10. 10.
    Dyerberg, J., Bang, H.O., and Hjorne, N. (1975) Fatty Acid Composition of the Plasma Lipids in Greenland Eskimos, Am. J. Clin. Nutr. 28, 958–966.PubMedGoogle Scholar
  11. 11.
    Thorngren, M., and Gustafson, A. (1981) Effects of 11-Week Increases in Dietary Eicosapentaenoic Acid on Bleeding Time, Lipids, and Platelet Aggregation, Lancet 2, 1190–1193.PubMedCrossRefGoogle Scholar
  12. 12.
    Kromhout, D., Bosschieter, E.B., and de Lezenne Coulander, C. (1985) The Inverse Relation Between Fish Consumption 20-Year Mortality from Coronary Heart Disease. N. Engl. J. Med. 312, 1205–1209.PubMedGoogle Scholar
  13. 13.
    Ascherrio, A., Rimm, E.B., Stampfer, M.J., Giovannucci, E.L., and Willett, W.C. (1995) Dietary Intake of Marine n-3 Fatty Acids, Fish Intake, and the Risk of Coronary Disease Among Men, N Engl. J. Med. 332, 977–982.CrossRefGoogle Scholar
  14. 14.
    Daviglus, M.L., Stamler, J., Orencia, A.J., Dyer, A.R., Liu, K., Greenland, P., Walsh, M.K., Morris, D., and Shekelle, R.B. (1997) Fish Consumption and the 30-Year Risk of Fatal Myocardial Infarction. N. Engl. J. Med. 336, 1046–1053.PubMedCrossRefGoogle Scholar
  15. 15.
    Fernandez, E., Chatenoud, L., La Vecchia, C., Negri, E., and Franceschi, S. (1999) Fish Consumption and Cancer Risk, Am. J. Clin. Nutr. 70, 85–90.PubMedGoogle Scholar
  16. 16.
    Cheng, J., Ogawa, K., Kuriki, K., Yokoyama, Y., Kamiya, T., Seno, K., Okuyama, H., Wang, J., Luo, C., Fujii, T., et al., (2003) Increased Intake of n-3 Polyunsaturated Fatty Acids Elevates the Level of Apoptosis in the Normal Sigmoid Colon of Patients Polypectomized for Adenomas/Tumors, Cancer Lett. 193, 17–24.PubMedCrossRefGoogle Scholar
  17. 17.
    Mori, T.A., Burke, V., Puddey, I.B., Shaw, J.E., and Beilin, L.J. (2004) Effect of Fish Diets and Weight Loss on Serum Leptin Concentration in Overweight, Treated-Hypertensive Subjects. J. Hypertens. 22, 1983–1990.PubMedCrossRefGoogle Scholar
  18. 18.
    Wakai, K., Tamakoshi, K., Date, C., Fukui, M., Suzuki, S., Lin, Y., Niwa, Y., Nishio, K., Yatsuya, H., Kondo, T., et al., for the JACC Study Group. (2005) Dietary Intakes of Fat and Fatty Acids and Risk of Breast Cancer: A Prospective Study in Japan, Cancer Sci. 96, 590–599.PubMedCrossRefGoogle Scholar
  19. 19.
    Kojima, M., Wakai, K., Tokudome, S., Suzuki, K., Tamakoshi, K., Watanabe, Y., Kawado, M., Hashimoto, S., Hayakawa, N., Ozasa, K., et al., for the JACC Study Group. (2005) Serum Levels of Polyunsaturated Fatty Acids and Risk of Colorectal Cancer: A Prospective Study, Am. J. Epidemiol. 161, 462–471.PubMedCrossRefGoogle Scholar
  20. 20.
    Kuriki, K., Wakai, K., Hirose, K. Matsuo, K., Ito, H., Suzuki, T., Saito, T., Kanemitsu, Y., Hirai, T., Kato, T., et al. (in press) Risk of Colorectal Cancer Is Linked to Erythrocyte Compositions of Fatty Acids as Biomarkers for Dietary Intakes of Fish, Fat and Fatty Acids.Google Scholar
  21. 21.
    Pala, V., Krogh, V., Muti, P., Chajes, V., Riboli, E., Micheli, A., Saadatian, M., Sieri, S., and Berrino, F. (2001) Erythrocyte Membrane Fatty Acids and Subsequent Breast Cancer: A Prospective Italian Study, J. Natl. Cancer Inst. 93, 1088–1095.PubMedCrossRefGoogle Scholar
  22. 22.
    Tiemeier, H., van Tuijl, H.R., Hofman, A., Kiliaan, A.J., and Breteler, M.M. (2003) Plasma Fatty Acid Composition and Depression Are Associated in the Elderly: The Rotterdam Study, Am. J. Clin. Nutr. 78, 40–46.PubMedGoogle Scholar
  23. 23.
    Wang, L., Folsom, A.R., Zheng, Z.J., Pankow, J.S., and Eckfeldt, J.H., for the ARIC Study Investigators. (2003) Plasma Fatty Acid Composition and Incidence of Diabetes in Middle-Aged Adults: The Atherosclerosis Risk in Communities (ARIC) Study, Am. J. Clin. Nutr. 78, 91–98.PubMedGoogle Scholar
  24. 24.
    Hunter, D. (1990) Biochemical Indicators of Dietary Intake in Nutritional Epidemiology (Willett, W., ed.), pp. 186–216, Oxford University Press, New York.Google Scholar
  25. 25.
    Lands, W.E.M. (1995) Long-Term Fat Intake and Biomarkers, Am. J. Clin. Nutr. 61 (Suppl), 721S-725S.PubMedGoogle Scholar
  26. 26.
    Adersen, L.F., Solvoll, K., and Drevon, C.A. (1996) Very-Long-Chain n-3 Fatty Acids as Biomarkers for Intake of Fish and n-3 Fatty Acids Concentrates, Am. J. Clin. Nutr. 64, 301–311.Google Scholar
  27. 27.
    Arab, L. (2003) Biomarkers of Fat and Fatty Acid Intake, J. Nutr. 133 (Suppl3), 925S-932S.PubMedGoogle Scholar
  28. 28.
    Kobayashi, M., Sasaki, S., Kawabata, T., Hasegawa, K., Akabane, M., and Tsugane, S. (2001) Single Measurement of Serum Phospholipid Fatty Acid as a Biomarker of Specific Fatty Acid Intake in Middle-Aged Japanese Men, Eur. J. Clin. Nutr. 55, 643–650.PubMedCrossRefGoogle Scholar
  29. 29.
    Kuriki, K., Nagaya, T., Imaeda, N., Tokudome, Y., Fujiwara, N., Sato, J., Ikeda, M., Maki, S., and Tokudome, S. (2002) Discrepancies in Dietary Intakes and Plasma Concentrations of Fatty Acids According to Age Among Japanese Female Dietitians, Eur. J. Clin. Nutr. 56, 524–531.PubMedCrossRefGoogle Scholar
  30. 30.
    Kuriki, K., Nagaya, T., Tokudome, Y., Imaeda, N., Fujiwara, N., Sato, J., Goto, C., Ikeda, M., Maki, S., Tajima, K., and Tokudome, S. (2003) Plasma Concentrations of (n-3) Highly Unsaturated Fatty Acids Intakes: A Cross-Sectional Study, J. Nutr. 133, 3643–3650.PubMedGoogle Scholar
  31. 31.
    Wakai, K., Ito, Y., Kojima, M., Tokudome, S., Ozasa, K., Inaba, Y., Yagyu, K., and Tamakoshi, A., JACC Study Group (2005) Intake Frequency of Fish and Serum Levels of Long-Chain n-3 Fatty Acids: A Cross-Sectional Study Within the Japan Collaborative Cohort Study, J. Epidemiol. 15, 211–218.PubMedCrossRefGoogle Scholar
  32. 32.
    Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957) A Simple Method for the Isolation and Purification of Total Lipides from Animal Tissues, J. Biol. Chem. 226, 497–509.PubMedGoogle Scholar
  33. 33.
    Skipski, V.P., Good, J.J., Barclay, M., and Reggio, R.B. (1968) Quantitative Analysis of Simple Lipid Classes by Thin-Layer Chromatography. Biochim Biophys Acta. 152, 10–19.PubMedGoogle Scholar
  34. 34.
    Kim, H.Y., and Salem, N. (1990) Separation of Lipid Classes by Solid Phase Extraction. J. Lipid Res. 31, 2285–2289.PubMedGoogle Scholar
  35. 35.
    Morrison, W.R., and Smith, L.M. (1964) Preparation of Fatty Acid Methyl Esters and Dimethylacetals from Lipids with Boron Fluoride-Methanol, J. Lipid Res. 5, 600–608.PubMedGoogle Scholar
  36. 36.
    Lepage, G., and Roy, C.C. (1986) Direct Transesterification of All Classes of Lipids in a One-Step Reaction. J. Lipid Res. 27, 114–120.PubMedGoogle Scholar
  37. 37.
    Ohta, A., Mayo, M.C., Kramer, N., and Lands, W.E. (1990) Rapid Analysis of Fatty Acids in Plasma Lipids, Lipids 25, 742–747.PubMedCrossRefGoogle Scholar
  38. 38.
    Christie, W.W. (1998) Gas Chromatography-Mass Spectrometry Methods for Structural Analysis of Fatty Acids, Lipids 33, 345–353.CrossRefGoogle Scholar
  39. 39.
    Masood, A., Stark, K.D., and Salem, N., Jr. (2005) A Simplified and Efficient Method for the Analysis of Fatty Acid Methyl Esters Suitable for Large Clinical Studies. J. Lipid Res. 46, 2299–2305.PubMedCrossRefGoogle Scholar
  40. 40.
    Snyder, J.M., King, J.W., and Jackson, M.A. (1996) Fat Content for Nutritional Labeling by Supercritical Fluid Extraction and an On-Line Lipase Catalyzed Reaction, J. Chromatogr A. 750, 201–207.PubMedCrossRefGoogle Scholar
  41. 41.
    Tokudome, S., Yokoyama, Y., Kamiya, T., Seno, K., Okuyama, H., Kuriki, K., Cheng, J., Nakamura, T., Fujii, T., Ichikawa, H., and Itoh, M. (2002) Rationale and Study Design of Dietary Intervention in Patients Polypectomized for Tumors of the Colorectum, Jpn. J. Clin. Oncol. 32, 550–553.PubMedCrossRefGoogle Scholar
  42. 42.
    Tokudome, S., Imaeda, N., Tokudome, Y. Fujiwara, N., Nagaya, T., Sato, J., Kuriki, K., Ikeda, M., and Maki, S. (2001) Relative Validity of a Semi-Quantitative Food Frequency Questionnaire Versus 28 Day Weighed Diet Records in Japanese Female Dietitians, Eur J Clin Nutr. 55, 735–742.PubMedCrossRefGoogle Scholar
  43. 43.
    Tokudome, Y., Kuriki, K., Imaeda, N., Ikeda, M., Nagaya, T., Fujiwara, N., Sato, J., Goto, C., Kikuchi, S., Maki, S., and Tokudome, S. (2003) Seasonal Variation in Consumption and Plasma Concentrations of Fatty Acids in Japanese Female Dietitians, Eur. J. Epidemiol. 18, 945–953.PubMedCrossRefGoogle Scholar
  44. 44.
    Jiang, J., Suzuki, S., Xiang, J., Kuriki, K., Hosono, A., Arakawa, K., Wang, J., Nagaya, T., Kojima, M., Katsuda, N., and Tokudome, S. (2005) Plasma Carotenoid, Alpha-Tocopherol and Retinol Concentrations and Risk of Colorectal Adenomas: A Case-Control Study in Japan, Cancer Lett. 226, 133–141.PubMedCrossRefGoogle Scholar
  45. 45.
    Tajima, K., Hirose, K., Inoue, M., Takezaki, T., Hamajima, N., and Kuroishi, T. (2000) A Model of Practical Cancer Prevention for Out-Patients Visiting a Hospital: The Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HER-PACC), Asian Pac. J. Cancer Prev. 1, 35–47.PubMedGoogle Scholar
  46. 46.
    Nagaya, Y (1994) Relationships Between Serum Saturated Fatty Acids and Serum Total Cholesterol and HDL-Cholesterol in Humans, Ann. Clin. Biochem. 31, 240–244.PubMedGoogle Scholar
  47. 47.
    Ozawa, A., Takayanagi, K., Fujita, T., Hirai, A., Hamazaki, T., Terano, T., Tamura, Y., and Kumagai, A. (1982) Determination of Long Chain Fatty Acids in Human Total Plasma Lipids Using Gas Chromatography, Bunseki-kagaku 31, 87–91 (in Japanese).Google Scholar
  48. 48.
    Dodge, J.T., Mitchell, C., and Hanahan, D.J. (1963) The Preparation and Chemical Characteristics of Hemoglobin-Free Ghosts of Human Erythrocytes, Arch. Biochem. Biophys. 100, 119–130.PubMedCrossRefGoogle Scholar

Copyright information

© AOCS Press 2006

Authors and Affiliations

  • Kiyonori Kuriki
    • 1
  • Kazuo Tajima
    • 1
  • Shinkan Tokudome
    • 2
  1. 1.Division of Epidemiology and Prevention, Aichi Cancer Center Research InstituteNagoyaJapan
  2. 2.Department of Health Promotion and Preventive MedicineNagoya City University Graduate School of Medical SciencesNagoyaJapan

Personalised recommendations